Percentage of Selected News Items and Attitude

Para a quantificação das proteínas totais utilizou-se o kit DC “Protein ssay” (Biorad, Hercules, California, EUA), baseado no método de Lowry modificado. Utilizou-se como padrão para a curva de calibração albumina de soro bovino ultrapura (Biorad).

4.4.3. Western blot

O método de western blot foi utilizado para a avaliação dos níveis de expressão da proteína Pgp. Foram utilizados extractos das linhas celulares MDA-MB 231, MCF-7,

71 MCF-7 Pgp. Foram também analisados extractos proteicos dos tumores de animais inoculados com células das MDA-MB 231, MCF-7, MCF-7 Pgp, em paralelo com extractos proteicos obtidos a partir de alíquotas das células inoculadas.

Alíquotas de extractos proteicos das células (100 g) e dos lisados tumorais (50 g) foram sujeitas a electroforese num gel 7% de SDS-poliacrilamida . Os géis foram posteriormente transferidos para uma membrana de nitrocelulose (Semi-Dry Transfer UN – TE 70 PWR – Amersham - Biosciences). A membrana foi incubada à temperatura ambiente com agitação 50 rpm com uma solução de PBS suplementado com 0,1 % (v/v) Tween20 (PBS-T), contendo 5 % de leite magro em pó, com o objectivo de bloquear os locais não específicos de ligação. Após este processo, a membrana foi então incubada com os anticorpos - primários específicos para a Pgp (1:500, monoclonal de ratinho, C219, Abcam, Cambridge, Reino Unido) e para a actina (1:15000, monoclonal de ratinho, AC-40, Sigma Aldrich), durante a noite a 4 ºC. No dia seguinte as membranas foram lavadas com PBS-T e incubadas durante 40 min, com o anticorpo secundário (1:3000,IgG-HRP de cabra, Biorad). As membranas foram de seguida incubadas com o kit “SuperSignal WestPico Substrate” (Thermo Scientific) seguindo as especificações do produto, para a detecção do sinal em chapas radiográficas.

73

CAPÍTULO 5

75

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